Summary

While Wnt/β-catenin signaling is known to be involved in the
development of neural crest cells in zebrafish, it is unclear which Wnts are
involved, and when they are required. To address these issues we employed a
zebrafish line that was transgenic for an inducible inhibitor of
Wnt/β-catenin signaling, and inhibited endogenous Wnt/β-catenin
signaling at discrete times in development. Using this approach, we defined a
critical period for Wnt signaling in the initial induction of neural crest,
which is distinct from the later period of development when pigment cells are
specified from neural crest. Blocking Wnt signaling during this early period
interfered with neural crest formation without blocking development of dorsal
spinal neurons. Transplantation experiments suggest that neural crest
precursors must directly transduce a Wnt signal. With regard to identifying
which endogenous Wnt is responsible for this initial critical period, we
established that wnt8 is expressed in the appropriate time and place
to participate in this process. Supporting a role for Wnt8, blocking its
function with antisense morpholino oligonucleotides eliminates initial
expression of neural crest markers. Taken together, these results demonstrate
that Wnt signals are critical for the initial induction of zebrafish neural
crest and suggest that this signaling pathway plays reiterated roles in its
development.

The Wnt family of secreted signaling molecules, which has been shown to
modulate cell proliferation, fate, and behavior in both vertebrates and
invertebrates (reviewed in Cadigan and
Nusse, 1997), has multiple roles in neural crest development
(reviewed by Dorsky et al.,
2000a; Yanfeng et al.,
2003). Several studies have implicated Wnts in neural crest
formation. Overexpression of various Wnts induces ectopic expression of neural
crest markers in Xenopus neuralized animal caps and embryos, while
expression of dominant-negative Wnts represses neural crest markers
(Saint-Jeannet et al., 1997;
Chang et al., 1998; Labonne and
Bronner-Fraser, 1998, Bang et
al., 1999; Tan et al.,
2001; Villanueva et al.,
2002). In addition, it was recently shown that inhibition of Wnt
signaling in vivo blocks expression of avian neural crest markers, and that
addition of soluble Wnt is sufficient to induce neural crest from neural tube
explants (García-Castro et al.,
2002). It should be noted that none of these studies identified
which specific Wnt molecules are required at particular stages of development
for neural crest induction or maintenance. Mice null for Wnt1 and Wnt3A show a
loss of neural crest derived cell types, including peripheral sensory neurons
and pigment cells (Ikeya et al.,
1997). However, these ligands are expressed after the initial
appearance of neural crest, and expression of markers for pre-migratory neural
crest are not changed in mutant mice. These results suggest that if a Wnt
signal is needed for initial neural crest induction, another Wnt ligand must
be involved. Wnt signals are involved in early patterning of the embryo,
imparting posterior character on neuroectoderm
(McGrew et al., 1995;
McGrew et al., 1997;
Bang et al., 1997;
Lekven et al., 2001;
Erter et al., 2001;
Kiecker and Niehrs, 2001) and
ventral character on mesoderm (Christian et al., 1993;
Hoppler et al., 1996;
Marom et al., 1999). It is
thus not clear whether Wnt-mediated effects on neural crest induction are
direct or indirect.

There are several lines of evidence suggesting that Wnt/β-catenin
signaling is also involved later in neural crest cell fate specification after
crest cells have been formed. Activation of Wnt/β-catenin signaling in
pre-migratory zebrafish neural crest cells promotes pigment cell determination
at the expense of neurons and glia, while inhibition of the Wnt/β-catenin
signaling pathway promotes neuronal and glial cell fates
(Dorsky et al., 1998). These
data support a model in which Wnt signaling is required for the pigment cell
lineage. However, they do not identify which Wnts are involved in this fate
decision. As noted, Wnt1/Wnt3A knockout mice are deficient in several neural
crest derivatives, including pigment cells
(Ikeya et al., 1997), but this
is probably due to lack of early expansion rather than effects on later cell
fate decisions. In addition, it has recently been shown that Wnts influence
the development of crest-derived pigment cells in chicks
(Jin et al., 2001), and that
Wnt/β-catenin signaling is necessary for mouse pigment cell
differentiation (Hari et al.,
2002). Wnts may also promote the proliferative expansion of
melanocyte precursors as well as promote their differentiation
(Dunn et al., 2000;
Yasumoto et al., 2002).
Together these studies suggest that Wnts are used at sequential stages of
neural crest development, both initially in crest induction and subsequently
in cell fate determination.

In this study we address how reiterated Wnt signaling influences neural
crest development in zebrafish. By inducing expression of a Wnt/β-catenin
signaling pathway inhibitor, we identify specific stages of development during
which Wnt/β-catenin mediated signaling is required cell-autonomously for
neural crest induction. We also identify a specific Wnt, Wnt8, and demonstrate
that it is crucial for initial neural crest induction. What emerges from our
data is the concept of a reiterated Wnt/β-catenin signaling mechanism.
The Wnt/β-catenin signaling pathway, probably in conjunction with other
secreted factors such as BMPs and FGFs, functions early to induce the
pre-migratory neural crest cells. Later, secreted Wnt signals provide
environmental cues during crest cell migration to specify which cells will
adopt the pigment cell fate.

Materials and methods

Fish maintenance and transgenic fish

Fish were maintained as described
(Westerfield, 1994). Wild-type
fish used were of the AB strain. The stable transgenic line carrying the Wnt
pathway inhibitor was generated as follows. The dominant-negative T-cell
Factor 3 reporter construct (hsΔTcf-GFP) was generated by replacing the
N-terminus of zebrafish Tcf3a (Pelegri and
Maischein, 1998; Dorsky et
al., 1999) with GFP and placing the fusion construct under the
control of the zebrafish hsp70 promoter
(Halloran et al., 2000).
N-terminally truncated Tcf3 is a dominant repressor of Wnt-mediated
transcription (Molenaar et al.,
1996). The linearized plasmid for this construct was injected into
1-2 cell zebrafish embryos. These fish were reared to adulthood. Adult
siblings were inter-crossed to identify founders with stable genomic
integration of the transgene (Tg (hsp70:ΔTCF-GFP)w26). Transgenic
TOPdGFP fish (Tg (TOP:dGFP)w25) express d2EGFP under control of four Lef
binding sites and have been described previously
(Dorsky et al., 2002).

Morpholino injections

Anti-sense Morpholino oligonucleotides (GeneTools) were dissolved in 1X
Danieau's buffer (Nasevicius and Ekker,
2000) for a stock concentration of 20 ng/nl. Morpholino
oligonucleotides (MOs) used in this study have the same sequences as wnt8 MO1
and MO2 (Lekven et al., 2001).
Each MO was injected into embryos at the 1-2 cell stage using an ASI pressure
injector (ASI Systems).

Antibody staining

To detect Foxd3 expression, fixed embryos were stained with anti-Foxd3
rabbit antisera (1:1000) with 20% goat serum, and then incubated with
anti-rabbit Alexa568-conjugated secondary antibodies. To detect Hu expression,
fixed embryos were stained with mouse monoclonal anti-Hu antibody (Molecular
Probes; 1:1000) with 10% goat serum, followed by incubation with anti-mouse
Alexa568-conjugated secondary antibody. All incubations were performed either
at room temperature for 4 hours or at 4°C for 12-16 hours. Following
antibody staining, serial washes in PBS+0.1% TritonX-100 were performed to
reduce background. Staining was visualized with a Zeiss LSM 510 Pascal
confocal microscope.

Transplantation assays

Donor embryos were labeled at the 1-4 cell stage with rhodamine-dextran.
Cells from blastula stage donor embryos were transplanted to mediolateral
regions of shield-stage, unlabeled host embryos
(Moens and Fritz, 1999). After
transplantation, embryos were placed in embryos medium
(Westerfield, 1994) containing
50 units penicillin and 5 μg streptomycin at 28.5°C. To activate
expression of the hsΔTcf-GFP, embryo incubation temperature was shifted
to 37°C for 1 hour. Transplanted cells were visualized using a Zeiss
compound microscope.

Results

Expression of ΔTcf inhibits expression of Wnt targets

To identify where and when Wnt/β-catenin signals might be involved in
zebrafish neural crest induction, we generated a transgenic line that allowed
conditional expression of a truncated form of the zebrafish tcf3 gene
headless (Pelegri and Maischein,
1998; Dorsky et al.,
1999; Kim et al.,
2000). We replaced the N-terminal domain of TCF with GFP,
eliminating the β-catenin binding domain and producing a dominant
inhibitor of Wnt signaling (Molenaar et
al., 1996). The fusion gene was then placed under control of the
zebrafish hsp70 promoter
(Halloran et al., 2000). To
confirm that this hs-ΔTcf construct can affect Wnt signaling in vivo, we
tested its ability to block a well-characterized action of Wnt8 in zebrafish:
ventrolateral mesoderm patterning (Lekven
et al., 2001; Erter et al.,
2001). The expression of the homeobox gene gsc is
restricted to the dorsal region of the marginal zone by post-MBT Wnt signaling
prior to gastrulation (Laurent et al.,
1997). Activating the hs-ΔTcf transgene at 4hpf resulted in
radial gsc expression 2 hours later at shield stage, as expected if
it inhibited Wnt8 (Fig. 1B,
compared with control, A). This indicates that global heat shock-induced
expression of ΔTcf functions is expected to antagonize Wnt signaling
during mesodermal patterning, as shown previously
(Pelegri and Maischein,
1998).

To determine whether hs-ΔTcf represses transcription of Wnt targets
in vivo, ΔTcf was induced by heat shock in fish that were transgenic for
TOPdGFP, a Wnt-responsive reporter construct that contains a destabilized
variant GFP under the control of four Lef binding sites
(Dorsky et al., 2002). Since
the destabilized GFP reporter has different codon usage than the GFP tag used
in the ΔTcf construct, its expression can be specifically detected by
in-situ hybridization. At 24hpf, embryos were exposed to heat shock for 1 hour
at 37°C, and then sorted into two pools, those expressing ΔTcf and
control siblings not expressing ΔTcf, based on GFP expression. The GFP
associated with hs-ΔTcf is ubiquitously expressed at very high levels
and localized in cell nuclei, which is readily distinguishable from TOPdGFP,
which is fainter, cytoplasmic, and has a restricted expression domain. At 2 or
6 hours after heat shock, TOPdGFP is expressed in the brain and spinal cord of
control embryos (Fig. 1C,E). In
contrast, TOPdGFP is visibly reduced by 2 hours after heat shock inΔ
Tcf-expressing embryos, and is further attenuated at 6 hours after heat
shock (Fig. 1D,F), suggesting
that ΔTcf remains active over this time. Consistent with these
observations, nuclear GFP expression in hs-ΔTcf embryos remains visible
at 6 hours after heat shock (data not shown). These results indicate that upon
rapid induction of ΔTcf expression, the transgene quickly represses
target gene transcription in a manner detectable by in-situ hybridization, and
this repression is maintained for at least 6 hours following heat shock
treatment.

Canonical Wnt signaling is required for neural crest induction

If Wnt signals are indeed necessary for zebrafish neural crest induction,
then inhibition of Wnt/β-catenin mediated signaling should result in loss
of neural crest. We globally blocked Wnt signaling at various stages of early
neural development by activating expression of ΔTcf in transgenic fish
(Fig. 2). Embryos were
incubated at 37°C for 45 minutes, and then returned to 28.5°C before
fixing and staining with an antibody that recognizes zebrafish Foxd3 (fkd6)
(Odenthal and Nusslein-Volhard,
1998), revealing neural crest cells flanking the neural plate. At
this stage, foxd3 appears to be expressed in virtually all
pre-migratory neural crest cells, completely overlapping with
snail2/slug (Kelsh et al.,
2000). Wnt/β-catenin signaling is crucial for neural crest
induction from the end of gastrulation (bud stage) through the 3-somite stage
since ΔTcf inhibits expression of Foxd3 at these stages
(Fig. 2D,E, compared with
control Fig. 2A,B). In
contrast, Foxd3 expression is comparable to that of wild-type siblings upon
expression of the inhibitor 1 hour later at the 6-somite stage
(Fig. 2C, compared with
control, Fig. 2F). Expression
of the neural crest marker sox10 as assayed by in-situ hybridization
is similarly affected (data not shown). We conclude that there is a temporal
limit to the requirement for Wnt/β-catenin mediated signaling in neural
crest induction, and that cells lose sensitivity to transgene activation
between the 3- and 6-somite stages.

Temporal limit for the Wnt/β-catenin signaling requirement.
Foxd3-positive neural crest cells, identified by anti-Foxd3 primary antibody
(red), flank the neural plate after heat activation at bud (A), 3-somite (B),
and 6-somite stages (C). Transgenic embryos show significant loss of Foxd3
expression upon heat activation of hs-ΔTcf (green) (D,E), while Foxd3
expression is comparable to that of wild-type siblings after induction at
6-somites (F). All images are dorsal views, anterior to top, of stacked 10X
confocal series after fixation at 3-somites (A,D) or 10-somites (B,C,E,F).
Expression of the transgene is shown in the insets (D,E,F).

To assess the effect of ΔTcf expression on the dorsal/ventral
patterning of the neural tube, we assayed expression of a pan-neuronal marker,
Hu (Marusich et al., 1994). Hu
protein is expressed in the trigeminal ganglia, primary motor neurons and
dorsal Rohon-Beard sensory neurons (Kim et
al., 1996). While the global loss of Wnt/β-catenin signaling
in response to ΔTcf results in loss of Foxd3 expression
(Fig. 3B, compared with control
Fig. 3A), Hu expression is
unaffected in transgenic embryos (Fig.
3D) or their wild-type siblings
(Fig. 3C) after heat-activation
at the 3-somite stage. Notably, differentiation of dorsal Rohon-Beard sensory
neurons (white arrow and arrowhead) and ventral motor neurons (asterisk) of
the spinal cord is unaffected. Hu expression is found in the same cells that
express the transgene (Fig. 3E,
yellow cells). These results suggest that the requirement for
Wnt/β-catenin mediated signaling at this stage is specific for neural
crest and not indirectly through its role in overall patterning of
neuroectoderm.

Loss of canonical Wnt signaling does not alter Hu expression. (A,B)
Foxd3-positive neural crest cells (outline empty arrowhead), identified by
anti-Foxd3 primary antibody (red), flank the neural plate after heat
activation at the 3-somite stage. As shown in (B), transgenic embryos show
significant loss of Foxd3 expression upon heat activation of hs-ΔTcf
(green) as compared to with wild-type siblings (A). (C,D,E) Anti-Hu antibody,
which recognizes the pan-neuronal marker Hu, was were used to identify
Hu-positive cells in heat-activation treated embryos (red). Normal expression
is detected in the trigeminal ganglia (white arrowhead), Rohon-Beard sensory
neurons (white arrow), and primary motorneurons in the spinal column
(asterisk) in ΔTcf embryos (D) as compared to with their wild-type
siblings (C). Overlap between Hu and transgene expression is seen at higher
power (E). All images are dorsal views of flat-mounted embryos fixed at the
10-somite stage, anterior to top, of stacked confocal series obtained at 10X
(A-D) or 20X (E).

The requirement for canonical Wnt signaling in the induction of neural
crest may be cell-autonomous, in which neural crest precursors must directly
receive a Wnt signal and activate expression of Wnt target genes to establish
their identity. Alternatively, the requirement may be non-autonomous; for
example, neural crest precursors might be induced by other signals derived
from the neural plate after this tissue has received posteriorizing Wnt
signals. To distinguish between these two possible scenarios, we tested the
ability of transplanted neural crest precursors to rescue the loss of neural
crest in ΔTcf transgenics. Small numbers of cells were transplanted into
the neural crest fate map position at shield stage. As a control, cells were
transplanted from wild-type donors into wild-type host embryos. The
transplantation process does not affect expression of Foxd3 in transplanted
neural crest precursors (Fig.
4A and inset). If the requirement for Wnt response were
cell-autonomous, transgenic neural crest precursors will be unable to express
Foxd3 in wild-type embryos after activation of the ΔTcf transgene at bud
stage. Consistent with this hypothesis, ΔTcf transgenic cells were
unable to form neural crest cells in wild-type hosts, although cells were
intercalated between wild-type cells expressing Foxd3
(Fig. 4B,C). In reciprocal
transplants, we observed Foxd3-positive nuclei derived from wild-type
transplanted cells in the ΔTcf transgenic host background
(Fig. 4D,E,F). As shown,
Foxd3-positive nuclei within the clone of cells derived from wild-type donors
do not co-localize with host ΔTcf-positive nuclei. Taken together, these
results suggest that cells require reception of a Wnt signal to be induced as
neural crest.

Canonical Wnt/β-catenin signaling is required in neural crest
precursors cell-autonomously. (A) As a control, rhodamine dextran-labeled
wild-type neural crest precursors (red) were transplanted into a wild-type
host embryo. The transplanted cells express Foxd3, detected with anti-Foxd3
antibody (green) when transplanted into a wild-type host embryo. Inset is a
single confocal slice to indicate co-localization in single transplanted cell
(white arrowhead). (B,C) Transgenic crest precursors fail to express Foxd3
when transplanted into a wild-type environment. Embryo shown is a dorsal view
with anterior to the top. Inset in (B) is magnified in (C). (D-F) Wild-type
cells express Foxd3 when transplanted into a transgenic environment. (D)
Foxd3-positive nuclei, detected with anti-Foxd3 primary antibody (red), are
observed in transplanted cells from wild-type donor embryos. (E) GFP-positive
nuclei (green) are observed in the ΔTcf background after heat-activation
of the transgene. (F) Stacked confocal images have been merged to show of
failure of Foxd3-positive nuclei to co-localize with background GFP
expression.

Wnt8 is involved in neural crest induction

The temporal requirement for canonical Wnt signaling during neural crest
development determined by the preceding data corresponds with the expression
of zebrafish wnt8. Zebrafish wnt8 is a bicistronic gene,
yielding two transcripts (wnt8.1 and wnt8.2) with
overlapping but distinct expression domains early in gastrulation
(Kelly et al., 1995;
Lekven et al., 2001). We
performed in-situ hybridization to determine the correspondence of
wnt8 to the earliest identified marker of the neural crest domain,
pax3. Both wnt8.1 and wnt8.2 transcripts are
expressed in proximity to, and partially overlapping with, pax3,
demonstrating that they may play a role in neural crest induction
(Fig. 5).

Wnt8 and pax3 show overlapping expression in the
presumptive neural crest domain. (A) Whole-mount in-situ hybridization at the
80% epiboly stage reveals expression of wnt8 transcripts at the
margin and along the dorsal side of the embryo. (B) Expression of
pax3 in the presumptive neural crest (arrow). (C) The wnt8
expression domain is in proximity to, and partially overlapping with,
expression of pax3 in the presumptive neural crest region (black
arrow). All images are lateral views, with dorsal to the right, of 80% epiboly
stage embryos after hybridization.

To test the importance of Wnt8 in the initial induction of neural crest, we
used antisense MOs to interfere with the translation of Wnt8.1 and 8.2.
Embryos were carefully staged by counting somites to discount any possible
delays in development after MO injection. Injection of MOs against
wnt8.1 at the 1-2 cell stage resulted in significant loss of
expression of pax3, foxd3 and sox10
(Fig. 6B,E,H, compared with
controls, Fig. 6A,D,G).
Expression of pax3 completely overlaps with foxd3 at this
stage, and in addition is expressed in some cells of the dorsal neural plate
(J.W.R., unpublished). sox10 expression is found in a subset of cells
that express foxd3 (Dutton et
al., 2001). This effect appears to be accompanied by lateral
expansion of the neural plate and disruption of the notochord, as indicated by
foxd3 expression in the notochord, consistent with previously
described roles for wnt8 in zebrafish development
(Erter et al., 2001;
Lekven et al., 2001). The loss
of early neural crest marker expression by wnt8.1 MO was
dose-dependent (data not shown), nearing 100% at 20 ng injected. In contrast,
blocking wnt8.2 function showed no loss of any of these markers at
all concentrations tested (Fig.
6C,F,I). Embryos co-injected with wnt8.1 MO and
wnt8.2 MO showed complete loss of neural crest marker expression and
similar A/P axis patterning defects, as previously described
(Lekven et al., 2001). These
data support a critical role for Wnt8 at early stages of neural crest
induction.

Wnt8 is required for neural crest induction in vivo. Whole-mount in-situ
hybridization at the 6-somite stage reveals loss of early neural crest markers
foxd3 (A-C), pax3 (D-F), and sox10 (G-I) upon
injection of 20 mg/ml wnt8.1 Morpholino oligonucleotides (B,E,H). Such loss
was not detected upon injection of 20 mg/ml wnt8.2 Morpholino (C,F,I),
suggesting it does not have a role in neural crest induction. Uninjected
controls (A,D,G) show normal expression of markers in the neural crest;
foxd3 is also expressed in the notochord. All images are dorsal
views, anterior to top, of 3-somite embryos.

To assess the requirement for Wnt8 in later specification of neural crest
derivatives, we again blocked Wnt8.1 function by injection of antisense MO and
assayed for the expression of various neural crest markers. Injection of 20 ng
wnt8.1 MO blocked neither expression of sox10 in
pre-migratory neural crest at the 10-somite stage nor sox10
expression in migratory crest at 24hpf
(Fig. 7B,D, compared with
uninjected controls, Fig.
7A,C). Similarly, injection of wnt8.1MO did not affect
either expression of mitfa, a melanophore-specific neural crest
marker (Fig. 7F, compared with
control, Fig. 7E) or
dlx2, a marker of cranial neural crest
(Fig. 7H, compared with
control, Fig. 7G)
(Akimenko et al., 1994). These
data suggest the loss of neural crest observed at the 3-somite stage upon
elimination of functional Wnt8 recovers by the 10-somite stage. This result is
consistent with the temporal limits for Wnt/β-catenin mediated signaling
established by the ΔTcf transgenic experiments. It is also consistent
with the restriction of wnt8 mRNA expression to the tailbud during
somitogenesis (Kelly et al.,
1995) (data not shown).

To test whether Wnt/β-catenin signaling is required for proper
specification of neural crest-derived cell types later in development, we used
the stable ΔTcf transgenics to block canonical Wnt/β-catenin
signaling at the onset of neural crest migration. Activation of the transgene
for 1 hour at 18hpf resulted in dramatic loss at 24hpf of mitfa, a
specific marker of crest-derived melanophores
(Lister et al., 1999),
compared with wild-type siblings (Fig.
8B, compared with control, Fig.
8A), consistent with previous results that mitfa is a
direct target of Wnt/β-catenin signaling
(Dorsky et al., 2000b). In
addition, expression of dlx2 in cranial neural crest cells migrating
into the developing branchial arches is substantially reduced upon transgene
activation (Fig. 8F,H, compared
with controls, Fig. 8E,G). In
contrast, transgene activation at this stage did not eliminate expression of
more broadly expressed markers of migrating crest, such as sox10
(Fig. 8D, compared with
control, Fig. 8C) and
crestin (not shown), although some reduction of sox10
expression was consistently observed. These data support a model where
Wnt/β-catenin mediated signaling is involved in specification of certain
neural crest lineages, including the pigment cell and possibly craniofacial
lineages, although Wnt signals no longer appear to be required for general
neural crest identity as cells migrate from the dorsal neural tube. This
Wnt/β-catenin requirement for melanophore and cranial crest specification
appears to be independent of the requirement for Wnt8 in early crest
induction.

Discussion

We present here evidence that Wnt signaling plays stage-specific roles
during neural crest induction in zebrafish. wnt8 is expressed at the
right time and place to be involved in the initial induction of neural crest,
and antisense MOs block induction. Using the hs-ΔTcf transgenic line, we
have identified a critical period during which neural crest formation is
disrupted that corresponds to the time when wnt8 perturbation affects
this process. Transplantation studies with this transgenic line demonstrate
that neural crest cells require the reception of the Wnt signal directly.
These studies confirm and extend previous findings demonstrating a specific
requirement for Wnt signaling in neural crest induction. Bang et al.
(Bang et al., 1999) previously
demonstrated that overexpression of Wnt8 induces expanded expression of
pax3 in Xenopus embryos. Additional studies have
demonstrated that misexpression of other Wnts, including wnt1, wnt3a
and wnt7b, induces expanded expression of Xenopus neural
crest markers (Saint-Jeannet et al.,
1997; Chang and
Hemmati-Brivanlou, 1998;
LaBonne and Bronner-Fraser,
1998; Villanueva et al.,
2002), and treatment of chick explants with soluble
Drosophila Wingless protein promotes neural crest formation
(García-Castro et al.,
2002). Together these studies have implicated a Wnt signal in
neural crest formation but did not specifically identify which ligand was
necessary.

Our studies are the first to identify a specific Wnt necessary for neural
crest induction in zebrafish. However, wnt8 is apparently needed only
during the initial phase of neural crest formation, and perhaps other Wnts are
needed after this period. In mouse embryos, loss of Wnt1 and
Wnt3a does not block the initial induction of neural crest but
affects subsequent neural crest expansion
(Ikeya et al., 1997). Although
Wnts are thought to be involved in regulating proliferation of neural tissue
(McMahon and Bradley, 1990;
Dickinson et al., 1994;
Megason and McMahon, 2002),
proliferation of neural crest precursors is reported to be unaffected in
Wnt1/Wnt3a knockout mice
(Ikeya et al., 1997). These
results suggest the possibility that there are several stages of neural crest
induction regulated by different Wnts: an early phase involving wnt8
and a later phase utilizing wnt1 and wnt3a. Other Wnts, such
as wnt3, wnt6, wnt7 and wnt10b, may also be involved.

Several studies have implicated Wnts in promoting ventral/posterior
patterning of mesoderm and neuroectoderm. Blocking wnt8 in zebrafish
results in loss of posterior brain and spinal cord
(Erter et al., 2001;
Lekven et al., 2001). In
addition, there is an anterior expansion of posterior markers when zebrafish
tcf3 genes are inactivated (Kim
et al., 2000; Dorsky et al.,
2003), including an anterior shift of neural crest
(Itoh et al., 2002). Since
relief of Tcf3 repressor activity is a result of Wnt signal transduction, loss
of tcf3 function is thought to behave similarly to Wnt activation
(Brannon et al., 1997;
Kim et al., 2000). Together
these observations suggest the possibility that the effects we see on neural
crest are indirect: interfering with Wnt signals might simply convert
posterior neural tissue to anterior, from which no neural crest would normally
be produced. However, our experiments with the ΔTcf transgenic line
suggest that this is not the case. We have found that neural crest development
can be blocked at a time when the development of dorsal spinal Rohon-Beard
sensory neurons is unaffected. This result implies that we have not simply
eliminated the domain from which neural crest cells are derived, since neural
crest cell precursors arise intermingled among Rohon-Beard neurons
(Cornell and Eisen, 2000). In
addition, cell transplantation experiments suggest that neural crest
precursors specifically require activation of the Wnt/β-catenin signaling
pathway, and that small groups of wild-type cells can form neural crest in the
correct position even when Wnt signaling is blocked in all other cells of the
host embryo. These results suggest that Wnt-mediated induction of zebrafish
neural crest is probably independent of its roles in anterior/posterior
patterning, as has been recently suggested for FGF-mediated induction of
neural crest in Xenopus
(Monsoro-Burq et al.,
2003).

At first glance, the phenotype of the headless/tcf3 mutant, in
which neural crest markers are expanded anteriorly, appears to contradict our
results with the ΔTcf transgenic line, in which neural crest markers are
lost. However, the headless mutation results in a loss-of-function of
a repressor while the ΔTcf transgenic results in a gain-of-function of a
repressor, which is consistent with the different phenotypes. Furthermore,
describing neural crest cells as `expanded' in headless mutants is
perhaps inaccurate, as the neural crest domain instead is shifted anteriorly.
Finally, the ΔTcf transgene is likely to act as a dominant repressor in
the place of all members of the TCF/Lef family, not just Tcf3.

There are several caveats to the interpretations of the data we present
here. Although wnt8 is expressed at the right place and time to be
involved in the induction of neural crest cells from ectoderm and
wnt8 MO injection blocks neural crest formation, we cannot eliminate
the possibility that activation of the ΔTcf transgene is instead
blocking signaling from some other Wnt. The period we identified as critical
using the transgenic line roughly corresponds to the period of wnt8
expression; however, exactly when Wnts are needed cannot be easily determined
by transgene activation. Although it takes several hours to inactivate the
TOPdGFP reporter, suggesting that the Wnt requirement for neural crest
formation might be later than the period in which we performed the heat shock,
this multicopy reporter transgene may not respond with the same kinetics as
endogenous genes. Currently identified zebrafish Wnts known to be expressed in
the dorsal neural tube, such as wnt1, wnt3a and wnt10b, are
expressed too late to be involved in this initial phase of neural crest
induction, and a deletion eliminating zebrafish wnt1 and
wnt10b has little effect on neural crest
(Lekven at al., 2003). Future
studies identifying other zebrafish Wnt genes and their functions will be
needed to fully address this point. Another caveat is that the ΔTcf
transgene may additionally act to repress genes not regulated by Wnt signals.
Future studies using other tools to block Wnt signals, such as identifying and
eliminating Frizzled receptors expressed at the right place and time, or
expressing other reagents that interfere with Wnt signals such as
axin or kinase-dead gsk3, are needed.

Both BMP and Notch signaling have been implicated in neural crest induction
in zebrafish, but these signaling pathways may be required at different times
from Wnt signaling. Mutations in zebrafish bmp2b result in loss of
both neural crest and Rohon-Beard cells
(Barth et al., 1999;
Nguyen et al., 2000).
Disruption of Notch signaling also results in loss of neural crest but instead
concomitantly increases the number of Rohon-Beard cells
(Cornell and Eisen, 2000). In
contrast, blocking Wnt signals can interfere with neural crest without
affecting Rohon-Beard cells, suggesting that Wnts maintain a role independent
of Notch regulation.

Previous studies have implicated Wnt signaling in the specification and
differentiation of pigment cells from neural crest
(Dorsky et al., 1998;
Dunn et al., 2000;
Jin et al., 2001;
Hari et al., 2002), and that
mitfa, a gene encoding a bHLH transcription factor necessary and
sufficient for pigment cell formation, is a direct target of the Wnt pathway
(Dorsky et al., 2000b;
Takeda et al., 2000;
Widlund et al., 2002). We
demonstrate here that the Wnt requirement for mitfa expression is to
some degree temporally separable from Wnt regulation of neural crest
induction, suggesting that reiterated Wnt signaling plays sequential roles in
neural crest development. When the Δ-Tcf transgene is activated at the
18-somite stage, mitfa expression is almost completely eliminated. An
alternative explanation is that transgene activation at this stage
specifically blocked the formation of a subpopulation of melanogenic neural
crest cells, consistent with the observed reduction of sox10-positive
cells. However, our previous results demonstrated that zebrafish melanogenic
neural crest cells have already segregated from the neural tube by the
18-somite stage (Raible and Eisen,
1994; Raible and Eisen,
1996), suggesting this possibility is less likely.

Our results also suggest that Wnt signals promote dlx2 expression
in branchial arches. Unfortunately, the poor long-term survival of
heat-shocked transgenic animals has not let us assess the final effects of
transgene activation on cartilage differentiation. Although ectomesenchyme
that gives rise to craniofacial cartilages is traditionally thought of as
derived from neural crest, some studies suggest that these cells are a
distinct population (e.g. Dutton et al.,
2001). Although our results might suggest that Wnt signals are
needed for ectomesenchyme as well as other neural crest derivatives, the
inference that these cells thus have common origins should be reached with
caution since Wnt signals have widespread use during embryogenesis.

Acknowledgments

We thank D. Grunwald, M. Halpern, R. Kelsh, J. Odenthal and H. Seo for
probes. We thank K. Cooper for assistance with the transplantation assays and
J. Lister for comments on the manuscript. Research was supported by the
Developmental Biology Training Grant and MCB graduate program (J.L.L.), HHMI
(R.T.M.), ACS (R.I.D.), NIH GM65469 (R.T.M. and D.W.R.) and NS35833
(D.W.R.).

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